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Background Aims: Evaluation of the BD Stem Cell Enumeration Kit was conducted at four clinical sites with flow cytometry CD34(+) enumeration to assess agreement between two investigational methods: (i) the BD FACSCanto II and BD FACSCalibur systems and (ii) the predicate method (Beckman Coulter StemKit and StemTrol, Immunotech SAS, Beckman Coulter, Marseille Cedex 9, France)., Methods: Leftover and delinked specimens (n = 1032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow and leucopheresis and cord blood anticoagulated with citrate phosphate dextrose, anticoagulant citrate dextrose-solution A, heparin and ethylenediaminetetraacetate, alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing and analysis., Results: The mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (-2.81 to 4.31 ±7.1) and BD FACSCalibur (-2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared with the predicate for viable CD34(+), percentage of CD34(+) in CD45(+) and viable CD45(+) populations (or gates). Bias analyses of the distribution of the predicate low, mid and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34(+), percentage of CD34(+) in CD45(+) and viable CD45(+). Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R(2) > 0.92 for both investigational methods., Discussion: In conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34(+), percentage of viable CD34(+) in CD45(+) and absolute viable CD45(+) populations., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: Enumeration of CD34(+) cells in leukocyte-rich cell suspensions is important for clinical decision-making in stem cell transplantation. Single-platform flow cytometry assays offer the significant advantages of speed and reproducibility, and have therefore become the gold standard in stem cell enumeration. The clinical community has recently defined the need for stem cell enumeration kits that incorporate viability dyes. The purpose of this study was to evaluate a novel assay, BD Biosciences' (BD) stem cell enumeration kit (SCE kit(‡)), in relation to Beckman Coulter's (BC) commercially available BC Stem-Kit™., Methods: Fresh/freeze-thawed samples from leukapheresis, bone marrow and cord blood, and fresh normal/mobilized blood, were analyzed with both assays (simultaneous detection of side/forward scatter and three fluorescence signals) on two flow cytometry platforms, BD FACSCanto II and BD FACSCalibur. Results. Results from both assays were highly congruent, with an overall r(2) ≥ 0.99 (all specimen types included), a linear correlation across all CD34(+) cell frequencies and concentrations, and an almost ideal steepness of the trend line., Conclusions: Both assays functioned reliably. Being based on single-platform International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines and similar staining methods, both assays essentially come to identical results. For most specimen types, the viability of CD34(+) cells was equal to overall leukocyte viability. In summary, in the hands of an experienced technician, the BD™ SCE kit and the BC Stem-Kit are equivalent. The infrequent user might derive benefit from the fact that counting spheres are pre-pipetted into the Trucount tube for the SCE kit, making this assay less susceptible to pipetting inaccuracy.

Although the influence of microenvironmental "niche" on the function of a variety of stem cells is undisputed, the details of hematopoietic stem cell/niche interactions at the cellular and molecular level have sparked a continuous debate. We studied the microanatomic partitioning of transplanted normal and alpha4 integrin-deficient Lin-kit+ cells in trabecular and compact bone before and after irradiation and present robust quantitative data on both. We found that (1) the microanatomic distribution of normal highly enriched progenitor cells is random in nonirradiated recipients based on area distribution analyses, (2) in contrast, in irradiated hosts normal cells distribute preferentially near the endosteum, (3) the overall cell seeding efficiency was higher in trabecular versus compact bone both before and after irradiation, and (4) alpha4 integrin-deficient cells not only lodge with reduced overall efficiency confirming previous data, but fail to preferentially partition themselves into endosteal regions in irradiated hosts, as normal cells do. A similar phenotype was observed with cells rendered G(i)-protein signaling incompetent by pertussis toxin treatment, supporting an active stromal-derived factor 1 (SDF-1) gradient near endosteum after irradiation.

The directed migration of mature leukocytes to inflammatory sites and the lymphocyte trafficking in vivo are dependent on G protein-coupled receptors and delivered through pertussis toxin (Ptx)-sensitive Gi-protein signaling. In the present study, we explored the in vivo role of G-protein signaling on the redistribution or mobilization of hematopoietic stem/progenitor cells (HPCs). A single injection of Ptx in mice elicits a long-lasting leukocytosis and a progressive increase in circulating colony-forming unit-culture (CFU-C) and colony-forming unit spleen (CFU-S). We found that the prolonged effect is sustained by a continuous slow release of Ptx bound to red blood cells or other cells and is potentially enhanced by an indirect influence on cell proliferation. Plasma levels of certain cytokines (interleukin 6 [IL-6], granulocyte colony-stimulating factor [G-CSF]) increase days after Ptx treatment, but these are unlikely initiators of mobilization. In addition to normal mice, mice genetically deficient in monocyte chemotactic protein 1 (MCP-1), matrix metalloproteinase 9 (MMP-9), G-CSF receptor, beta2 integrins, or selectins responded to Ptx treatment, suggesting independence of Ptx-response from the expression of these molecules. Combined treatments of Ptx with anti-very late activation antigen (anti-VLA-4), uncovered potentially important insight in the interplay of chemokines/integrins, and the synergy of Ptx with G-CSF appeared to be dependent on MMP-9. As Ptx-mobilized kit+ cells display virtually no response to stromal-derived factor 1 (SDF-1) in vitro, our data suggest that disruption of CXCR4/SDF-1 signaling may be the underlying mechanism of Ptx-induced mobilization and indirectly reinforce the notion that active signaling through this pathway is required for continuous retention of cells within the bone marrow. Collectively, our data unveil a novel example of mobilization through pharmacologic modulation of signaling.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a 5-day course of granulocyte colony-stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor plerixafor is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was coadministered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization achieved by the VLA4 inhibitor and CXCR2 agonist combination in mice compared with currently approved HSPC mobilization methods, the combination represents an exciting potential strategy for clinical development in the future., Introduction In postnatal mammals, the vast majority of hematopoietic stem and progenitor cells (HSPCs) reside in the protective environment of the bone marrow (BM) (1, 2). Accordingly, only very few [...]

Background: Because of limitations of transportation imposed by the COVID-19 pandemic, current recommendation calls for cryopreservation of allogeneic stem cell transplants before patient conditioning. A single cell therapy laboratory was selected to function as the central cryopreservation hub for all European registry donor transplants intended for the Australian-Pacific region. We examined properties of these transplants to ascertain how quality is maintained.Methods: We analyzed 100 pandemic-related allogeneic mobilized blood-derived stem cell apheresis products generated at 30 collection sites throughout Europe, shipped to and cryopreserved at our center between April and November of 2020. Products were shipped in the cool, subsequently frozen with DMSO as cryoprotectant. Irrespective of origin, all products were frozen within the prescribed shelf-life of 72 h.Results: Prior to cryopreservation, viable stem cell and leukocyte count according to the collection site and our reference laboratory were highly concordant (r2 = 0.96 and 0.93, respectively) and viability was > 90% in all instances. Median nominal post-thaw recovery of viable CD34+ cells was 42%. Weakly associated with poorer CD34+ cell recovery was higher leukocyte concentration, but not time lag between apheresis or addition of cryopreservant, respectively, and start of freezing. The correlation between pre- and post-thaw CD34+ cell dose was high (r2 = 0.85), hence predictable. Neutrophil and platelet engraftment were prompt with no evidence of dose dependency within the range of administered cell doses (1.31-15.56 × 106 CD34+ cells/kg).Conclusions: General cryopreservation of allogeneic stem cell transplants is feasible. While more than half of the CD34+ cell content is lost, the remaining stem cells ensure timely engraftment. [ABSTRACT FROM AUTHOR]

Background: Autologous stem cell transplantation remains an integral treatment tool for certain childhood malignancies. In children, a central venous catheter is typically necessary to provide adequate flow rates for preparative apheresis. In this study, the feasibility and efficiency of collecting CD34+ cells via an indwelling Hickman catheter, preimplanted for chemotherapy, instead of placing an additional temporary central venous catheter was evaluated.Study Design and Methods: Forty-eight pediatric leukaphereses for autologous hematopoietic stem cell transplantation using Spectra Optia MNC, Version 3.0 were reviewed. We compared preimplanted Hickman catheters with a temporary Shaldon catheter, inserted for apheresis. Apheresis was considered successful if a dose of 2 × 106 CD34+ peripheral blood stem cells/kg BW was achieved.Results: In 43 (89.6%) of the 48 patients, a Hickman catheter was used for leukapheresis. Only 5 patients (10.4%) received a temporary Shaldon catheter. In both groups, apheresis was performed without apparent adverse reactions. The dose of collected CD34+ peripheral blood stem cells was 12.7 × 106 (range, 2.3-70.7 × 106 ) cells/kg BW in the Hickman group and 16.2 × 106 (range, 3.8-48.4 × 106 ) cells/kg BW in the Shaldon group, showing no statistically significant difference (p = 0.58). In both groups, the primary endpoint of a minimal CD34+ cell concentration of 2 × 106 cells/kg BW was achieved at a maximum of two leukapheresis sessions. Apheresis efficacy was further confirmed by the collection efficiency of 40.2% in the Hickman group and 27.8% in the Shaldon group (p = 0.32).Conclusion: These data indicate the reliable feasibility and efficacy of mobilized apheresis via an indwelling Hickman catheter. In light of this, the routine insertion of a dialysis catheter for the purpose of leukapheresis should be critically reconsidered. [ABSTRACT FROM AUTHOR]

Background: Excessive T-cell depletion (TCD) is a prerequisite for graft manufacturing in haploidentical stem cell (SC) transplantation by using either CD34 selection or direct TCD such as CD3/CD19 depletion.Study Design and Methods: To optimize graft composition we compared 1) direct or indirect TCD only, 2) a combination of CD3/CD19-depleted with CD34-selected grafts, or 3) TCD twice for depletion improvement based on our 10-year experience with 320 separations in graft manufacturing and quality control.Results: SC recovery was significantly higher (85%, n = 187 vs. 73%, n = 115; p < 0.0001), but TCD was inferior (median log depletion, -3.6 vs. -5.2) for CD3/CD19 depletion compared to CD34 selection, respectively. For end products with less than -2.5 log TCD, a second depletion step led to a successful improvement in TCD. Thawing of grafts showed a high viability and recovery of SCs, but low NK-cell yield. To optimize individualized graft engineering, a calculator was developed to estimate the results of the final graft based on the content of CD34+ and CD3+ cells in the leukapheresis product.Conclusion: Finally, calculated splitting of the starting product followed by CD3/19 depletion together with CD34+ graft manipulation may enable the composition of optimized grafts with high CD34+-cell and minimal T-cell content. [ABSTRACT FROM AUTHOR]

Background: Automation of cell therapy manufacturing promises higher productivity of cell factories, more economical use of highly-trained (and costly) manufacturing staff, facilitation of processes requiring manufacturing steps at inconvenient hours, improved consistency of processing steps and other benefits. One of the most broadly disseminated engineered cell therapy products is immunomagnetically selected CD34+ hematopoietic "stem" cells (HSCs).Methods: As the clinical GMP-compliant automat CliniMACS Prodigy is being programmed to perform ever more complex sequential manufacturing steps, we developed a CD34+ selection module for comparison with the standard semi-automatic CD34 "normal scale" selection process on CliniMACS Plus, applicable for 600 × 10(6) target cells out of 60 × 10(9) total cells. Three split-validation processings with healthy donor G-CSF-mobilized apheresis products were performed; feasibility, time consumption and product quality were assessed.Results: All processes proceeded uneventfully. Prodigy runs took about 1 h longer than CliniMACS Plus runs, albeit with markedly less hands-on operator time and therefore also suitable for less experienced operators. Recovery of target cells was the same for both technologies. Although impurities, specifically T- and B-cells, were 5 ± 1.6-fold and 4 ± 0.4-fold higher in the Prodigy products (p = ns and p = 0.013 for T and B cell depletion, respectively), T cell contents per kg of a virtual recipient receiving 4 × 10(6) CD34+ cells/kg was below 10 × 10(3)/kg even in the worst Prodigy product and thus more than fivefold below the specification of CD34+ selected mismatched-donor stem cell products. The products' theoretical clinical usability is thus confirmed.Conclusions: This split validation exercise of a relatively short and simple process exemplifies the potential of automatic cell manufacturing. Automation will further gain in attractiveness when applied to more complex processes, requiring frequent interventions or handling at unfavourable working hours, such as re-targeting of T-cells. [ABSTRACT FROM AUTHOR]

Bone marrow mononuclear cells (BMCs) are suitable for bone tissue engineering. Comparative data regarding the needs of BMC for the adhesion on biomaterials and biocompatibility to various biomaterials are lacking to a large extent. Therefore, we evaluated whether a surface coating would enhance BMC adhesion and analyze the biocompatibility of three different kinds of biomaterials. BMCs were purified from human bone marrow aspirate samples. Beta tricalcium phosphate (β-TCP, without coating or coated with fibronectin or human plasma), demineralized bone matrix (DBM), and bovine cancellous bone (BS) were assessed. Seeding efficacy on β-TCP was 95% regardless of the surface coating. BMC demonstrated a significantly increased initial adhesion on DBM and β-TCP compared to BS. On day 14, metabolic activity was significantly increased in BMC seeded on DBM in comparison to BMC seeded on BS. Likewise increased VEGF-synthesis was observed on day 2 in BMC seeded on DBM when compared to BMC seeded on BS. The seeding efficacy of BMC on uncoated biomaterials is generally high although there are differences between these biomaterials. Beta-TCP and DBM were similar and both superior to BS, suggesting either as suitable materials for spatial restriction of BMC used for regenerative medicine purposes in vivo. [ABSTRACT FROM AUTHOR]

Biosimilars are approved biologics with comparable quality, safety, and efficacy to a reference product. Unlike generics, which are chemically manufactured copies of small-molecule drugs with relatively simple chemical structures, the biosimilar designation is applied to drugs that are produced by living organisms, implying much more difficult to control manufacturing and purification procedures. To account for these complexities, the European Medicines Agency (EMA), the US Food and Drug Administration, the Australian Therapeutic Goods Administration, and other regulatory authorities have devised and implemented specific, markedly more demanding pathways for the evaluation and approval of biosimilars. To date, several biosimilars have been approved, including versions of somatropin, erythropoietin, and granulocyte–colony-stimulating factor (G-CSF), and several biosimilar monoclonal antibodies are currently in development. The reference G-CSF product (Neupogen, Amgen) has been used for many years for prevention and treatment of neutropenia and also for mobilization of peripheral blood stem cells (PBSCs). However, concerns have been raised about the safety and efficacy of biosimilar G-CSF during PBSC mobilization procedures, especially in healthy donors. This article reviews the available evidence on the use of biosimilar G-CSF in this setting. Aggregate clinical evidence supports the assessment by the EMA of biosimilar and originator G-CSF as highly biologically similar, with respect to desired and undesired effects. [ABSTRACT FROM AUTHOR]

Human embryonic stem cells provide an alternative to using human embryos for studying developmentally regulated gene expression. The co-expression of high levels of embryonic ε and fetal γ globin by the hESC-derived erythroblasts allows the interrogation of ε globin regulation at the transcriptional and epigenetic level which could only be attained previously by studying cell lines or transgenic mice. In this study, we compared the histone modifications across the β globin locus of the undifferentiated hESCs and hESC-, FL-, and mobilized PB CD34 cells-derived erythroblasts, which have distinct globin expression patterns corresponding to their developmental stages. We demonstrated that the histone codes employed by the β globin locus are conserved throughout development. Furthermore, in spite of the close proximity of the ε globin promoter, as compared to the β or γ globin promoter, with the LCR, a chromatin loop was also formed between the LCR and the active ε globin promoter, similar to the loop that forms between the β or γ globin promoters and the LCR, in contrary to the previously proposed tracking mechanism. [ABSTRACT FROM AUTHOR]

BACKGROUND: Granulocyte-colony-stimulating factor-mobilized peripheral blood stem cells, collected by white blood cell apheresis, are used for more than 80% of allogeneic and most autologous hematopoietic stem cell transplantations. Optimal donor and recipient outcomes require maximized stem cell collection efficiency and minimized non-target cell contamination. Therefore, improved apheresis technology is desirable. The safety and feasibility of apheresis collections with the novel, electronics-assisted apheresis system Spectra Optia v.5.0 (CaridianBCT) were recently demonstrated. An unpublished optimization trial had furthermore determined that different settings than manufacturer-installed default might result in improved apheresis yields. STUDY DESIGN AND METHODS: The first prospective comparison of allogeneic peripheral blood stem cell apheresis with the Spectra Optia versus the COBE Spectra (CaridianBCT) mononuclear cell (MNC) in a routine clinical setting is reported here; 'optimized' machine settings were used. Assessed variables included collection efficiency, product characteristics, donor outcomes, and frequency of operator interventions. Outcomes were additionally compared with historical data from the Spectra Optia in default mode. RESULTS: The mean CD34+ cell collection efficiency CE1 was 7.9% greater with the Spectra Optia than with the COBE Spectra MNC. Variability of outcomes was equally great. Reduced platelet (PLT) attrition necessitated 90% fewer autologous PLT reinfusions. Spectra Optia products contained 50% fewer red blood cells, but 50% more granulocytic lineage cells. Less operator input was required, although 26% of Spectra Optia apheresis procedures required triggering of the first chamber flush. Apheresis yield and collection efficiency were also markedly greater than in default-mode Spectra Optia collections. CONCLUSION: Using optimized machine settings, peripheral blood stem cell apheresis outcomes with the automated apheresis system Spectra Optia exceed results with the COBE Spectra MNC or the Spectra Optia in the default mode. [ABSTRACT FROM AUTHOR]

Cryopreserved hematopoietic progenitor cells collected by apheresis from granulocyte-colony-stimulating factor with or without chemotherapy-mobilized patients have become the preferred type of autograft to support treatment of diseases amenable to high-dose chemotherapy. A novel apheresis system, the Spectra Optia v.5.0 (CaridianBCT), was constructed to meet certain shortcomings of manual apheresis systems such as the COBE Spectra MNC (CaridianBCT), including the need for continuous optical or manual monitoring and readjustment of buffy coat position and sensitivity to inconsistent blood flow. By use of optical sensors, which provide real-time automatic interface (buffy coat) and collection line control, the Spectra Optia promises to automatically guide apheresis procedures, potentially freeing up operator time and reducing variability in collection efficiency (CE2). In a two-center clinical trial, 35 autologous stem cell donors were subjected to apheresis with the Spectra Optia to validate feasibility and effectiveness of apheresis procedures. Results were compared to data from 80 autologous apheresis procedures with the COBE Spectra MNC. Usability and function of the automatic interface management were excellent. CD34+ cell quality, assessed by viability staining, colony-forming unit-culture frequency, and engraftment kinetics, was equally good with both systems. CE2 of the Spectra Optia, calculated as CD34+ contents in the product divided by the number of CD34+ cells presented to the collection port, exceeded that of the COBE Spectra MNC. Spectra Optia product volumes were significantly smaller. Very high white blood cell and platelet counts modestly reduced CE2 with the Spectra Optia. The Spectra Optia is a novel automatic apheresis system supporting autologous stem cell collection with at least equal efficiency and superior user-friendliness compared to the COBE Spectra MNC. [ABSTRACT FROM AUTHOR]

The article offers information on the continuous gene contact of stem cells. It states that the development and survival as well as the special functions of hematopoietic stem cells (HSCs) are placed on the bone marrow (BM). It cites the study conducted by Ryan and colleagues regarding the use of genetic approach to offer new understanding in the mobilization mechanism. It also states that this approach requires additional caution before it can be considered for cancer treatment

Background aims. Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective. Methods. We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immunomagnetically isolating them via the transiently up-regulated activation marker, CD 137. Results. In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From 2 x 109 peripheral blood mononuclear cells, we isolated 27 x 10³-318 x 10³ Aspergillus-specific T-helper cells. Frequency among total T cells was increased, on average, by 200-fold. Specific studies indicate specificity and functionality: After non-specific in vitro expansion and re-stimulation with different antigens, we observed strong cytokine responses to A. fumigatus and some other fungi including Candida albicans, but none to unrelated antigens. Discussion. Our technology isolates naturally occurring Aspergillus-specific T-helper cells within 2 days of identifying the clinical indication. Rapid adoptive transfer of Aspergillus-spccific T cells may be quite feasible; the clinical benefit remains to be demonstrated. A manufacturing license as an advanced-therapy medicinal product was received and a clinical trial in post-transplantation invasive aspergillosis patients approved. The product is dosed at 5 x 10E3/kg T cells (single intravenous injection), of which at least 10% must be A. fumigatus-specific. [ABSTRACT FROM AUTHOR]

BACKGROUND AIMS: Evaluation of the BD Stem Cell Enumeration Kit was conducted at four clinical sites with flow cytometry CD34+ enumeration to assess agreement between two investigational methods: (i) the BD FACSCanto II and BD FACSCalibur systems and (ii) the predicate method (Beckman Coulter StemKit and StemTrol, Immunotech SAS, Beckman Coulter, Marseille Cedex 9, France). METHODS: Leftover and delinked specimens (n = 1032) from clinical flow cytometry testing were analyzed on the BD FACSCanto II (n = 918) and BD FACSCalibur (n = 905) in normal and mobilized blood, frozen and thawed bone marrow and leucopheresis and cord blood anticoagulated with citrate phosphate dextrose, anticoagulant citrate dextrose-solution A, heparin and ethylenediaminetetraacetate, alone or in combination. Fresh leucopheresis analysis addressed site equivalency for sample preparation, testing and analysis. RESULTS: The mean relative bias showed agreement within predefined parameters for the BD FACSCanto II (-2.81 to 4.31 ±7.1) and BD FACSCalibur (-2.69 to 5.2 ±7.9). Results are reported as absolute and relative differences compared with the predicate for viable CD34+, percentage of CD34+ in CD45+ and viable CD45+ populations (or gates). Bias analyses of the distribution of the predicate low, mid and high bin values were done using BD FACSCanto II optimal gating and BD FACSCalibur manual gating for viable CD34+, percentage of CD34+ in CD45+ and viable CD45+. Bias results from both investigational methods show agreement. Deming regression analyses showed a linear relationship with R² > 0.92 for both investigational methods. DISCUSSION: In conclusion, the results from both investigational methods demonstrated agreement and equivalence with the predicate method for enumeration of absolute viable CD34+, percentage of viable CD34+ in CD45+ and absolute viable CD45+ populations. [ABSTRACT FROM AUTHOR]

Recently, new advances were made regarding the depletion of CD45RA+ naïve T cells from haploidentical grafts as they are suspected to be the most alloreactive. Within this project we investigated CD45RA-depletion from G-CSF mobilized PBSC by two different purification strategies according to GMP, specifically direct depletion of CD45RA+ cells (one-step approach), or CD34-positive selection followed by CD45RA-depletion (two-step approach). With log −3.9 and − 3.8 the depletion quality of CD45RA+ T cells was equally for both approaches together with a close to complete CD19+ B cell depletion. However, due to a high expression of CD45RA the majority of NK cells were lost within both CD45RA depletion strategies. Stem cell recovery after one-step CD45RA-depletion was at median 52.0% (range: 49.7–67.2%), which was comparable to previously published recovery data received from direct CD34 positive selection. Memory T cell recovery including CD4+ and CD8+ memory T cell subsets was statistically not differing between both purification approaches. The recovery of CD4+ and CD8+ T cells was as well similar, but overall a higher amount of cytotoxic than T-helper cells were lost as indicated by an increase of the CD4/CD8 ratio. CD45RA-depletion from G-CSF mobilized PBSC is feasible as one- and two-step approach and results in sufficient reduction of CD45RA+ T cells as well as B cells, but also to a co-depletion of NK cells. However, by gaining two independent cell products, the two-step approach enables the highest clinical flexibility in regard to individual graft composition with precise dosage of stem cells and T cells. [ABSTRACT FROM AUTHOR]